Detection of an antigen or an antibody by a colloidal gold immunoassay method is known for many years. The principle of this assay method is summarized as set out next. Namely, when a target substance is an antigen, for example, a colloidal gold which has been sensitized with an antibody capable of recognizing this antigen, that is, an antibody-sensitized colloidal gold is used in a reaction mixture containing, in general, the antigen and impurities and the like in an intimately mixed state, whereby the antibody-sensitized colloidal gold is specifically caused to bind to the antigen. A complex-containing reaction mixture, which contains a complex formed as a result of the reaction, is subjected to permeation like chromatographic development through a reaction mixture permeable material or to filtering permeation through a reaction mixture permeable material while making use of a diffusive infiltration phenomenon of the reaction mixture, so that the reaction mixture containing the complex, the antibody-sensitized colloidal gold, which has not reacted, and the impurities and the like are allowed to infiltratively move.
Further, by a capturing antibody which has been immobilized beforehand on the reaction mixture permeable material at a specific position on a moving path, binds specifically to the antigen and is different in antigen recognition site from the antibody-sensitized colloidal gold, the complex alone is captured at the specific position and the remaining matters are moved away along with the reaction mixture. By a color tone of the colloidal gold in the complex as appeared at the specific position, the antigen alone is distinguished or detected.
When the target is an antibody, on the other hand, a colloidal gold which has been sensitized with an antigen capable of recognizing the antibody (antigen-sensitized colloidal gold) is also reacted with the antibody by specific binding in a reaction mixture containing the antibody. Subsequently, the complex-containing reaction mixture which contains the resulting complex is caused to move through a reaction mixture permeable material by making use of a penetrative infiltration phenomenon as in the above-described detection. By either a capturing antibody or a capturing antigen which has been immobilized beforehand on the reaction mixture permeable material at a specific position and binds specifically to the antigen or antibody in the complex, only the complex out of the complex-containing reaction mixture is captured at the specific position. By a color tone of the colloidal gold in the complex as appeared at the specific position, the target antibody alone can be distinguished or detected.
In this colloidal gold immunoassay method, the specific binding is generally binding through an antigen-antibody reaction, and a usable colloidal gold is generally of the red type or the purple type.
The reaction mixture generally contains, as a medium, water which in turn contains biocomponents, buffer and the like. Water may be replaced in part by an inert water-soluble organic solvent such as dimethyl sulfoxide or dioxane with a view to assisting dissolution of the target substance in such a liquid-form medium, namely, liquid medium without interfering with the specific binding reaction. Further, an inert high-molecular substance may be added to protect the antibody or the like or to lower the dielectric constant of the reaction mixture containing the colloidal gold. Moreover, a surface active substance or the like can also be added for assisting the diffusive infiltration phenomenon of the reaction mixture.
As the reaction mixture permeable material, a sheet-like or laminate-like material equipped with permeability for the reaction mixture can be used. The target substance moves along with the reaction mixture by diffusive infiltration through interstices and pores in the material. This material is formed of at least one of sheets in the form of filter paper, cotton-like, sponge-like or porous films, and the like. The target substance is allowed to move generally in a coplanar direction in the case of a sheet-like material or in the direction of thickness in the case of a laminate-like material.
When the target substance in the colloidal gold immunoassay method is an antigen, it is general to use two types of antibodies for the antigen, to sensitize one of the antibodies, which is a monoclonal in general, with a colloidal gold into an antibody-sensitized colloidal gold, and to use the other antibody (either a monoclonal or a polyclonal), which is different in antigen recognition site from the antibody-sensitized colloidal gold, as a capturing antibody.
The colloidal gold immunoassay method will next be described in more detail on the basis of an example as applied for the detection of human chorionic gonadotropin (hCG) from human urine. For example, a piece of filter paper is used as a reaction mixture permeable material. A colloidal gold, which has been sensitized with an anti-hCG antibody (an anti-hCG monoclonal antibody labeled with colloidal gold particles), is held in a re-dissolvable (re-elutable) form near an end of the filter paper, whereas an anti-hCG polyclonal antibody or a monoclonal antibody different in antigen epitope from the above-described monoclonal antibody is immobilized as a capturing antibody in a form free from re-dissolution near an opposite end of the filter paper.
If needed for the confirmation of an end of development, a certain antibody for the above-described anti-hCG monoclonal antibody (for example, an anti-mouse IgG antibody if the monoclonal antibody is mouse IgG) is also immobilized at a position farther than a final end of the development as viewed in the direction of the development.
An assay is conducted by applying human urine, which contains the target substance, to the one end and then allowing the antigen and impurities to be developed along with the urine toward the opposite end by the diffusive infiltration phenomenon of the urine, which contains the antigen and the impurities and the like, in a similar manner as in paper chromatography.
By the development, the colloidal gold sensitized with the anti-hCG monoclonal antibody, said colloidal gold having been held near the one end, is first eluted into the urine. The colloidal gold and hCG, which is contained in the urine, forms an hCG-anti-hCG monoclonal antibody sensitized colloidal gold complex, which diffuses further toward the opposite end. This complex is captured by an antigen-antibody reaction with the immobilized capturing antibody, so that at the immobilized position of the capturing antibody, a color tone of the colloidal gold appears in an immobilized pattern (in the form of a spot, mark, letter or the like), thereby indicating the existence of hCG as the antigen in the urine.
On the other hand, the colloidal gold sensitized with the anti-hCG monoclonal antibody, which has been simply eluted in the urine and has not reacted with hCG, also diffuses along with the urine toward the opposite end. The unrealized conjugation is not ambushed behind antigen-antibody reaction with the capturing antibody and hence passes through the position of the capturing antibody. It is however captured by the anti-mouse IgG antibody immobilized near the final end, so that the color tone of the colloidal gold appears in an immobilized pattern (in the form of a spot, mark, letter or the like), thereby indicating that the development by the urine has been surely effected.
The colloidal gold immunoassay method is practiced under such a principle as described above and is used as a simple and easy assay method for substances in the body. This assay method however involves a problem in that the development of a color tone of a colloidal gold, said color tone being indicative of the results of a specific reaction for the detection of a target substance, is not always clear. There is hence a long standing desire for the development of a method which permits easy and clear recognition of a position colored with the colloidal gold without making darker a background color tone corresponding to a non-specific reaction.